ReviewActivity, function, and gene regulation of the catalytic subunit of telomerase (hTERT)
Introduction
The role of telomerase, the ribonucleoprotein responsible for maintaining the ends of chromosomes, has been the subject of intense investigation in recent years due to its potential role in cellular aging, cancer, and immortalization (Counter et al., 1992, Kim et al., 1994, Harley et al., 1990). In adult somatic cells, the ends of chromosomes contain tandem telomeric repeats (e.g. 5′-TTAGGG-3′ in humans) (Greider, 1996) that act to preserve chromosomal integrity by preventing degradation, end-to-end fusions, rearrangements, and chromosome attrition (Greider, 1991). In normal somatic cells, each cell division is associated with the loss of 30–150 bp of telomeric DNA (Vaziri et al., 1993, Harley et al., 1990, Allsopp et al., 1992). This loss of non-coding DNA at the ends of chromosomes eventually results in the reduction to a certain critical length associated with growth arrest and cellular senescence (Chiu and Harley, 1997, Autexier and Greider, 1996) (Fig. 1). In the presence of telomerase, an RNA-dependent DNA polymerase (Greider and Blackburn, 1987, Morin, 1989), telomere lengths are extended or maintained and replicative senescence is avoided (Vaziri and Benchimol, 1998, Bodnar et al., 1998) (Fig. 1). While most normal human somatic cells exhibit no detectable telomerase activity, highly proliferative cells such as germline cells, trophoblasts, hematopoietic cells, endometrial cells, and up to 95% of cancer cells express telomerase to varying degrees (Kim et al., 1994, Counter et al., 1994a, Counter et al., 1994b, Counter et al., 1995, Shay and Bacchetti, 1997, Wright et al., 1996, Yasumoto et al., 1996, Broccoli et al., 1995, Härle-Bachor and Boukamp, 1996) (Fig. 1).
Control of telomerase activity has been widely studied and mechanisms for post-translational regulation by phosphorylation have been suggested (Li et al., 1998, Kharbanda et al., 2000). However, the identification of differentially expressed subunits of telomerase have indicated a major control point at the level of transcription. Studies of the telomerase enzyme complex have revealed the presence of two major subunits contributing to enzymatic activity: an RNA component (hTER) that serves as the template for the polymerase activity of this enzyme and a catalytic subunit with reverse transcriptase activity (hTERT) (Nakayama et al., 1998, Harrington et al., 1997, Feng et al., 1995). Both hTER and hTERT are necessary for reconstitution of telomerase activity in vitro (Weinrich et al., 1997, Beattie et al., 1998). While hTER is widely expressed in embryonic and somatic tissue, hTERT is tightly regulated and is not detectable in most somatic cells (Meyerson et al., 1997, Nakamura et al., 1997). hTERT mRNA expression temporally parallels changes in telomerase activity during cellular differentiation (Bestilny et al., 1996, Savoysky et al., 1996, Xu et al., 1999) and neoplastic transformation (Takakura et al., 1998, Wu et al., 1999a) (Fig. 1). Further support for the essential role of hTERT comes from recent studies showing that ectopic expression of hTERT is sufficient for restoring telomerase activity in a number of telomerase-negative cell lines, including foreskin fibroblasts, mammary epithelial cells, retinal pigment epithelial cells, and umbilical endothelial cells (Weinrich et al., 1997, Vaziri and Benchimol, 1998, Wen et al., 1998, Bodnar et al., 1998, Counter et al., 1998). Although the additional inactivation of the RB/p16 pathway is required for the hTERT-mediated immortalization of keratinocytes and mammary epithelial cells (Kiyono et al., 1998), the relationship between hTERT expression and the capacity for cellular immortalization is well established. Taken together, these findings point to the expression of hTERT as the rate-limiting step in telomerase activity and bring the study of hTERT gene expression to the forefront of telomerase regulation research.
Elucidation of the mechanisms governing hTERT expression will likely have wide ranging effects on the study and treatment of cancer and other age-related diseases. Approximately 85–95% of tumorigenic tissues express hTERT (Shay and Bacchetti, 1997), making it a valuable tool in cancer diagnosis. Moreover, in some of the most common and lethal cancers, including thyroid (Umbricht et al., 1997), breast (Poremba et al., 1998), cervical (Kyo et al., 1998, Shroyer et al., 1998, Iwasaka et al., 1998), and prostate cancer (Zhang et al., 1998), hTERT expression is detectable in the early stages of malignancy. Additionally, the quantification of in vivo hTERT levels holds promise for cancer prognosis as high telomerase activity has been correlated with a poor prognosis for a number of cancers, including neuroblastoma (Hiyama et al., 1995b, Hiyama et al., 1997), acute myelogenous leukemia (Xu et al., 1998), breast (Clark et al., 1997), and gastrointestinal cancers (Hiyama et al., 1995c, Okusa et al., 1998). Since hTERT appears to be linked to the early progression as well as the severity of cancer, telomerase/hTERT inhibitors are currently being investigated for their therapeutic potential. Recent work with inducible dominant-negative mutants of hTERT and antisense telomerase has led to a marked reduction of endogenous telomerase activity in tumor cell lines and eventual death of these cells in vitro (Kondo et al., 1998, Zhang et al., 1999, Hahn et al., 1999a). Additional studies involving hammerhead ribozymes have identified a ribozyme (13-ribozyme) that is capable of targeting the 5′-end of hTERT mRNA and strongly inhibiting telomerase activity (Yokoyama et al., 2000). Further understanding of the mechanisms involved in hTERT regulation are necessary for the development of anticancer therapies in vivo, as well as for the study of aging and the treatment of other age-related diseases.
Section snippets
Establishment of a consensus nucleotide numbering system for the hTERT regulatory region
Though recent studies have examined the alternate splicing of the hTERT transcript (Kilian et al., 1997, Ulaner et al., 1998, Ulaner et al., 2000), the search for mechanisms governing the regulated activity of telomerase has focused on the level of transcription. The sequencing and characterization of the hTERT promoter region has enabled direct study of the molecular mechanisms involved in the regulation of hTERT gene expression (Cong et al., 1999, Horikawa et al., 1999, Takakura et al., 1999,
The hTERT promoter region is rich in transcription factor binding sites
Recent studies of the hTERT 5′ gene regulatory region have identified both canonical and non-canonical motifs for the binding of numerous transcription factors (Table 1, Table 2). While the exact location and number of these motifs varies according to the particular study, the presence of sites for multiple activators and repressors suggests a complex system of regulation. Of the transcription factors known to upregulate hTERT, the oncoprotein c-Myc is the most widely studied, though roles for
Transcriptional control of hTERT by differential methylation
Analysis of the hTERT 5′ gene regulatory region revealed the presence of a CpG island and a high overall GC content (Cong et al., 1999, Horikawa et al., 1999, Takakura et al., 1999, Wick et al., 1999). This feature, combined with the presence of binding sites for methylation-sensitive transcription factors within the hTERT core promoter (Table 1), suggests a possible role for methylation in the regulation of hTERT gene expression. Methylation-mediated control of gene expression is most often
Conclusion
Recent reports addressing hTERT regulation present multiple and wide-ranging mechanisms potentially involved in the transcriptional control of hTERT. From the identification of activators and repressors that bind to the hTERT 5′ regulatory region to the role of CpG methylation and histone acetylation, an abundance of regulatory models have been suggested. Further work will be necessary to determine how hTERT expression and telomerase activity are regulated both in vitro and in vivo.
Currently,
Acknowledgements
We thank Joyce Haskell for critical reading of the manuscript and Jason Key for technical assistance in preparation of the figures. This work was supported in part by grants from the John A. Hartford Foundation, the Southeast Center for Excellence in Geriatric Medicine, the American Cancer Society, and the UAB Center for Aging, Department of Biology and Natural Sciences and Mathematics Graduate School.
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